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BioMed Research International
Volume 2017, Article ID 3018608, 12 pages
https://doi.org/10.1155/2017/3018608
Research Article

His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?

1Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
2Institute for Interdisciplinary Research and Technologies, 1421 Sofia, Bulgaria
3Institute of Information and Communication Technologies, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
4Faculty of Physics, Sofia University “St. Kliment Ohridski”, 1164 Sofia, Bulgaria
5Proteros Biostructures, 82152 Martinsried, Germany

Correspondence should be addressed to Genoveva Nacheva; gb.sab.12oib@avevoneg

Received 1 March 2017; Accepted 23 April 2017; Published 8 June 2017

Academic Editor: Rituraj Purohit

Copyright © 2017 Elena Krachmarova et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved when the proteins were expressed in E. coli cells and the tag-free proteins showed fully restored activity. To shed light on this phenomenon we performed molecular dynamics simulations. The latter showed that the tags interact with the receptor binding domains and the flexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFNγ receptor. We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site.